Spermatogenesis is a complex process of cell differentiation involving interactions between germ cells, Sertoli cells, and other somatic cells within the testis. One feature of this differentiative process is the appearance of several germ cell- specific constituents in a precise temporal sequence. Immunological and biochemical techniques have been used to characterize such constituents, both in the acrosome and on the cell surface. Central to these studies are methods for the purification of germ cells at defined stages of spermatogenesis by unit gravity sedimentation. Three areas of research have been pursued: (a) Monoclonal antibodies have been used to characterize germ cell constituents expressed during restricted periods of spermatogenesis. Antibody 1D4 recognizes a set of glycoconjugates that appear in the acrosome of early spermatids but are modified during the late haploid stages so that the determinant no longer is detected. Additional monoclonal antibodies have been prepared against presumptive surface constituents excised from two-dimensional polyacrylamide gels. (b) Conditions for the short term culture of spermatogenic cells in serum-free medium have been refined to facilitate metabolic studies and the development of in vitro functional assays. Both pachytene spermatocytes and round spermatids cultured for 1-2 days with Sertoli cell-conditioned medium (SCM) maintain elevated viabilities and ATP levels. The active fraction of SCM that improves germ cell viability is relatively heat-stable, is eliminated by trypsin proteolysis, and appears to be larger than most growth factors. Culture studies also have shown that round spermatids are more sensitive than pachytene spermatocytes to the toxic effects of retinoids. (c) Isolated spermatogenic cells in short-term cultures have been used to monitor changes in protein synthesis and glycosylation during germ cell differentiation. Germ cell constituents exhibiting both tissue and stage specificity are candidates for further studies exploring gene regulation and cell-cell interactions during spermatogenesis.